Method of manufacture of colloidal rod particles as nanobarcodes

ABSTRACT

A method is disclosed for the manufacture of colloidal rod particles as nanobarcodes. Template membranes for the deposition of materials are prepared using photolithographic techniques.

RELATED APPLICATIONS

[0001] This application is a Continuation of U.S. Utility applicationSer. No. 09/969,518, entitled “Method Of Manufacture Of Colloidal RodParticles As Nanobarcodes,” filed Oct. 2, 2001, which is aContinuation-in-Part of U.S. Utility application Ser. No. 09/677,203,entitled “Method of Manufacture of Colloidal Rod Particles as NanobarCodes,” filed Oct. 2, 2000, which is a Continuation-in-Part of U.S.Utility application Ser. No. 09/598,395, filed Jun. 20, 2000, entitled“Colloidal Rod Particles as Nanobar Codes.” The Ser. No. 09/598,395Application was filed claiming the benefit of the filing date of U.S.Provisional Application Serial No. 60/157,326, filed Oct. 1, 1999,entitled “Self Bar-coded Colloidal Metal Nanoparticles;” U.S.Provisional Application Serial No. 60/189,151, filed Mar. 14, 2000,entitled “Nanoscale Barcodes;” U.S. Provisional Application Serial No.60/190,247, filed Mar. 17, 2000, entitled “Colloidal Rod Particles asBarcodes;” and U.S. Provisional Application Serial No. 60/194,616, filedApr. 5, 2000, entitled “Nanobarcodes: Technology Platform forPhenotyping.” The Ser. No. 09/677,203 application was filed claiming thebenefit of the filing date of U.S. Provisional Application Serial No.60/212,167, filed Jun. 16, 2000, entitled “Techniques for MultipleParallel Nanobarcode Synthesis.” This application also claims thebenefit of the filing date of U.S. Provisional Application Serial No.60/237,322, filed Oct. 2, 2000, entitled “Methods for the Manufacture ofColloidal Rod Particles as Nanobar Codes,” and U.S. ProvisionalApplication Serial No. 60/285,017, filed Apr. 19, 2001, entitled “Methodof Manufacture of Colloidal Rod Particles.”

FIELD OF THE INVENTION

[0002] The present invention is directed to methods of manufacture ofnanoparticles and approaches for such manufacture. In certain preferredembodiments of the invention, the nanoparticles may be used to encodeinformation and thereby serve as molecular (or cellular) tags, labelsand substrates.

[0003] The membranes of the present invention may be used as templatesfor the synthesis of nanoparticles according to methods provided herein.The membranes include anodized alumina membranes, polycarbonatetrach-etched membranes, and membranes made using photolithographicmethods. Throughout this application, said membranes may beinterchangeably referred to as “porous membranes,” “porous templates”and “templates.” Nanoparticles may be formed within the pores bydeposition methods including, electrochemical deposition, sequentialchemical reaction, and chemical vapor deposition (CVD). Alternatively,the nanoparticles maybe directly manufactured using photolithographictechniques.

BACKGROUND OF THE INVENTION

[0004] The present invention relates to methods of manufacture ofsegmented particles and assemblies of differentiable particles (whichmay or may not be segmented). Without a doubt, there has been a paradigmchange in what is traditionally defined as bioanalytical chemistry. Amajor focus of these new technologies is to generate what could becalled “increased per volume information content”. This term encompassesseveral approaches, from reduction in the volume of sample required tocarry out an assay, to highly parallel measurements (“multiplexing”),such as those involving immobilized molecular arrays, to incorporationof second (or third) information channels, such as in 2-D gelelectrophoresis or CE-electrospray MS/MS.

[0005] Unfortunately, many of these seemingly revolutionary technologiesare limited by a reliance on relatively pedestrian materials, methods,and analyses. For example, development of DNA microarrays (“gene chips”)for analysis of gene expression and genotyping by Affymetrix, Incyte andsimilar companies has generated the wherewithal to immobilize up to20,000 different fragments or full-length pieces of DNA in aspatially-defined 1-cm² array. At the same time, however, the use ofthese chips in all cases requires hybridization of DNA in solution toDNA immobilized on a planar surface, which is marked both by a decreasein the efficiency of hybridization (especially for cDNA) and a fargreater degree of non-specific binding. It is unclear whether theseproblems can be completely overcome. Moreover, there is a general senseof disillusionment both about the cost of acquiring external technologyand the lead-time required to develop DNA arraying internally.

[0006] A second example of how groundbreaking can be slowed by inferiortools is in pharmaceutical discovery by combinatorial chemistry. At themoment, solution phase, 5-10 μm diameter latex beads are usedextensively as sites for molecular immobilization. Exploiting the widelyadopted “split and pool” strategy, libraries of upwards of 100,000compounds can be simply and rapidly generated. As a result, thebottleneck in drug discovery has shifted from synthesis to screening,and equally importantly, to compound identification, (i.e., whichcompound is on which bead?). Current approaches to the latter comprise“bead encoding”, whereby each synthetic step applied to a bead isrecorded by parallel addition of an organic “code” molecule; reading thecode allows the identity of the drug lead on the bead to be identified.Unfortunately, the “code reading” protocols are far from optimal: inmost every strategy, the code molecule must be cleaved from the bead andseparately analyzed by HPLC, mass spectrometry or other methods. Inother words, there is at present no way to identify potentiallyinteresting drug candidates by direct, rapid interrogation of the beadson which they reside, even though there are numerous screening protocolsin which such a capability would be desirable.

[0007] Two alternative technologies with potential relevance both tocombinatorial chemistry and genetic analysis involve “self-encodedbeads”, in which a spectrally identifiable bead substitutes for aspatially defined position. In the approach pioneered by Walt andco-workers, beads are chemically modified with a ratio of fluorescentdyes intended to uniquely identify the beads, which are then furthermodified with a unique chemistry (e.g. a different antibody or enzyme).The beads are then randomly dispersed on an etched fiber array so thatone bead associates with each fiber. The identity of the bead isascertained by its fluorescence readout, and the analyte is detected byfluorescence readout at the same fiber in a different spectral region.The seminal paper (Michael et al., Anal. Chem. 70, 1242-1248 (1998)) onthis topic points out that with 6 different dyes (15 combinations ofpairs) and with 10 different ratios of dyes, 150 “unique opticalsignatures” could be generated, each representing a different bead“flavor”. A very similar strategy is described by workers at Luminex,who combine flavored beads ready for chemical modification (100commercially available) with a flow cytometry-like analysis. (See, e.g.,McDade et al., Med. Rev. Diag. Indust. 19, 75-82 (1997)). Once again,the particle flavor is determined by fluorescence, and once thebiochemistry is put onto the bead, any spectrally distinct fluorescencegenerated due to the presence of analyte can be read out. Note that ascurrently configured, it is necessary to use one color of laser tointerrogate the particle flavor, and another, separate laser to excitethe bioassay fluorophores.

[0008] A more significant concern with self-encoded latex beads is thelimitations imposed by the wide bandwidth associated with molecularfluorescence. If the frequency space of molecular fluorescence is usedboth for encoding and for bioassay analysis, it is hard to imagine how,for example, up to 20,000 different flavors could be generated. Thisproblem might be alleviated somewhat by the use of combinations ofglass-coated quantum dots, which exhibit narrower fluorescencebandwidths. (See, e.g. Bruchez et al., Science, 281, 2013-2016 (1998)).However, these “designer” nanoparticles are quite difficult to prepare,and at the moment, there exist more types of fluorophores than(published) quantum dots. If, however, it were possible to generate verylarge numbers of intrinsically-differentiable particles by some means,then particle-based bioanalysis would become exceptionally attractive,insofar as a single technology platform could then be considered for themultiple high-information content research areas; includingcombinatorial chemistry, genomics, and proteomics (via multiplexedimmunoassays).

[0009] Previous work has originally taught how metal can be depositedinto the pores of a metallized membrane to make an array of metalnanoparticles embedded in the host. Their focus was on the opticaland/or electrochemical properties of these materials. A similartechnique was used to make segmented cylindrical magnetic nanoparticlesin a host membrane, where the composition of the particles was variedalong the length. In no case, however, have freestanding, rod-shapednanoparticles with variable compositions along their length beenprepared. Indeed, “freestanding” rod-shaped metal nanoparticles of asingle composition, in which the length is at least one micron, havenever been reported. Likewise, freestanding rod-shaped metalnanoparticles not embedded or otherwise contained within such hostmaterials have never been reported. See, Martin et al., Adv. Materials11:1021-25 (1999).

SUMMARY OF THE INVENTION

[0010] Rod-shaped nanoparticles have been prepared whose composition isvaried along the length of the rod. These particles are referred to asnanoparticles or nanobar codes, though in reality some or all dimensionsmay be in the micron size range. The present invention is directed tomethods of manufacture of such nanoparticles.

[0011] The present invention includes methods of manufacture offree-standing particles comprising a plurality of segments, wherein theparticle length is from 10 nm to 50 μm and particle width is from 5 nmto 50 μm. The segments of the particles of the present invention may becomprised of any material. Included among the possible materials are ametal, any metal chalcogenide, a metal oxide, a metal sulfide, a metalselenide, a metal telluride, a metal alloy, a metal nitride, a metalphosphide, a metal antimonide, a semiconductor, a semi-metal, anyorganic compound or material, any inorganic compound or material, aparticulate layer of material or a composite material. The segments ofthe particles of the present invention may be comprised of polymericmaterials, crystalline or non-crystalline materials, amorphous materialsor glasses. In certain preferred embodiments of the invention, theparticles are “functionalized” (e.g., have their surface coated with IgGantibody). Commonly, such functionalization may be attached on selectedor all segments, on the body or one or both tips of the particle. Thefunctionalization may actually coat segments or the entire particle.Such functionalization may include organic compounds, such as anantibody, an antibody fragment, or an oligonucleotide, inorganiccompounds, and combinations thereof. Such functionalization may also bea detectable tag or comprise a species that will bind a detectable tag.

[0012] Also included within the present invention are methods ofmanufacture of an assembly or collection of particles comprising aplurality of types of particles, wherein each particle is from 20 nm to50 μm in length and is comprised of a plurality of segments, and whereinthe types of particles are differentiable. In the preferred embodiments,the particle types are differentiable based on differences in thelength, width or shape of the particles and/or the number, composition,length or pattern of said segments. In other embodiments, the particlesare differentiable based on the nature of their functionalization orphysical properties (e.g., as measured by mass spectrometry or lightscattering).

[0013] The present invention includes the manufacture of nanobar codesby the electrochemical deposition of metals inside a template whereinthe process is improved, separately and collectively, by i)electroplating in an ultrasonication bath; and ii) controlling thetemperature of the deposition environment, preferably by using arecirculating temperature bath.

[0014] Also included within the scope of the invention are methods forthe simultaneous or parallel manufacture of a plurality of differenttypes of nanobar codes. According to one such method, a plurality oftemplates are held in a common solution chamber and electrochemicaldeposition is accomplished by controlling deposition at each membrane byapplying current selectively to predetermined electrodes associated witheach such membrane.

[0015] Also included within this invention is an apparatus for themanufacture of nanobar codes comprising: a plating solution cell, adefined-pore size template, means for applying a current to causeelectrochemical deposition of a metal into said template, means foragitation of the plating solution, such as an ultrasonic transducer, andtemperature control means.

[0016] Also included within this invention is an apparatus for thesimultaneous manufacture of a plurality of different types of nanobarcodes. In one embodiment, such apparatus comprises: a solution chamber,a plurality of templates, means for selectively applying a current toeach of said templates, and control means for operating said apparatus.

[0017] Also within the scope of the invention are methods of makingsegmented nanoparticles using a porous template manufactured by standardphotolithographic techniques, comprising exposing a pattern on aresist-coated substrate or multi-layer stack and then etching theexposed pattern to form pores.

[0018] Also included within the invention are methods for formingnanoparticles by exposing a pattern on a resist-coated substratecomprising one or more layers of metal, then etching the exposed patternto form free-standing nanoparticles.

BRIEF DESCRIPTION OF THE FIGURES

[0019]FIG. 1 is a perspective view of an apparatus for manufacturing aplurality of different types of nanobar codes.

[0020]FIG. 2 is a cross-sectional elevation view of the apparatus ofFIG. 1.

[0021]FIG. 3 is a schematic illustration of a four-layer stack on asilicon wafer substrate (A) before exposure; (B) following exposure anddevelopment of the photoresist, and etching to etch stop; (C) followingfurther etching to conductive layer, (D) after formation of thesegmented nanoparticle, and (E) the liberated segmented nanoparticles.

[0022]FIG. 4 is an SEM (top view) of a template prepared usingphotolithographic techniques in which nanoparticles have been formed byelectrochemical deposition. The pore diameter is approximately 2.5 to 3μm.

[0023]FIG. 5 is an SEM (side view) of a free-standing nanoparticle madeby electrodeposition in a template prepared using photolithographictechniques.

[0024]FIG. 6 is an SEM (cross-sectional view) of a template preparedusing photolithographic techniques.

DETAILED WRITTEN DESCRIPTION OF THE INVENTION

[0025] The present application is directed to methods of manufacture ofnanoparticles. Such nanoparticles and their uses are described in detailin U.S. Utility application Ser. No. 09/598,395, filed Jun. 20, 2000,entitled “Colloidal Rod Particles as Nanobar Codes,” incorporated herebyin its entirety by reference. Also incorporated herein in their entiretyby reference, are two U.S. Utility applications entitled “Methods ofImaging Colloidal Rod Particles as Nanobarcodes” and “Colloidal RodParticles as Nanobar Codes.” The present application is filed as aContinuation-in-Part of the Ser. No. 09/677,203 application.

[0026] The synthesis and characterization of multiple segmentedparticles is described in Martin et al., Adv. Materials 11: 1021-25(1999). The article is incorporated herein by reference in its entirety.Also incorporated herein by reference in their entirety are U.S.Provisional Application Serial No. 60/157,326, filed Oct. 1, 1999,entitled “Self Bar-coded Colloidal Metal Nanoparticles”; U.S.Provisional Application Serial No. 60/189,151, filed Mar. 14, 2000,entitled “Nanoscale Barcodes”; U.S. Provisional Application Serial No.60/190,247, filed Mar. 17, 2000, entitled “Colloidal Rod Particles asBarcodes”; U.S. Provisional Application Serial No. 60/194,616, filedApr. 5, 2000, entitled “Nanobarcodes: Technology Platform forPhenotyping;” U.S. Provisional Application Serial No. 60/237,322, filedOct. 2, 2000, entitled “Methods for the Manufacture of Colloidal RodParticles as Nanobar Codes;” and U.S. Provisional Application Serial No.60/285,017, filed Apr. 19, 2001, entitled “Method of Manufacture ofColloidal Rod Particles.”

[0027] Because bar coding is so widely-used in the macroscopic world,the concept has been translated to the molecular world in a variety offigurative manifestations. Thus, there are “bar codes” based on analysisof open reading frames, bar codes based on isotopic mass variations, barcodes based on strings of chemical or physical reporter beads, bar codesbased on electrophoretic patterns of restriction-enzyme cleaved mRNA,bar-coded surfaces for repeatable imaging of biological molecules usingscanning probe microscopies, and chromosomal bar codes (a.k.a.chromosome painting) produced by multi-chromophore fluorescence in situhybridization. All these methods comprise ways to code biologicalinformation, but none offer the range of advantages of the bonafide barcodes of the present invention, transformed to the nanometer scale.

[0028] The particles to be manufactured according to the presentinvention are alternately referred to as nanoparticles, nanobar codes,rods, nanorods, Nanobarcodes™ particles, and rod shaped particles. Tothe extent that any of these descriptions may be considered as limitingthe scope of the invention, the label applied should be ignored. Forexample, although in certain embodiments of the invention, theparticle's composition contains informational content, this is not truefor all embodiments of the invention. Likewise, although nanometer-sizedparticles fall within the scope of the invention, not all of theparticles of the invention fall within such size range.

[0029] In certain preferred embodiments of the present invention, thenanobar code particles are manufactured by electrochemical deposition inan alumina or polycarbonate template, followed by template dissolution,and typically, they are prepared by alternating electrochemicalreduction of metal ions, though they may easily be prepared by othermeans, both with or without a template material. Typically, the nanobarcodes have widths between 30 nm and 1,000 nanometers, though they canhave widths of several microns. Likewise, while the lengths (i.e. thelong dimension) of the materials are typically on the order of 1 to 15microns, they can easily be prepared in lengths as long as 50 microns,and in lengths as short as 20 nanometers. In some embodiments, thenanobar codes comprise two or more different materials alternated alongthe length, although in principle as many as dozens of differentmaterials could be used. Likewise, the segments could consist ofnon-metallic material, including but not limited to polymers, oxides,sulfides, semiconductors, insulators, plastics, and even thin (i.e.,monolayer) films of organic or inorganic species.

[0030] When the particles of the present invention are made byelectrochemical deposition, the length of the segments (as well as theirdensity and porosity) can be adjusted by controlling the amount ofcurrent (or electrochemical potential) passed in each electroplatingstep; as a result, the rod resembles a “bar code” on the nanometerscale, with each segment length (and identity) programmable in advance.Other forms of deposition can also yield the same results. For example,deposition can be accomplished via electroless processes and inelectrochemical deposition by controlling the area of the electrode, theheterogenous rate constant, the concentration of the plating material,and the potential and combinations thereof (collectively referred toherein as electrochemical deposition). The same result could be achievedusing another method of manufacture in which the length or otherattribute of the segments can be controlled. While the diameter of therods and the segment lengths are typically of nanometer dimensions, theoverall length is such that in preferred embodiments it can bevisualized directly in an optical microscope, exploiting thedifferential reflectivity of the metal components.

[0031] The particles of this embodiment of the present invention aredefined in part by their size and by the existence of at least 2segments. The length of the particles can be from 10 nm up to 50 μm. Inpreferred embodiments the particle is 500 nm-30 μm in length. In themost preferred embodiments, the length of the particles of thisinvention is 1-15 μm. The width, or diameter, of the particles of theinvention is within the range of 5 nm-50 μm. In preferred embodimentsthe width is 10 nm-1 μm, and in the most preferred embodiments the widthor cross-sectional dimension is 30 nm-500 nm.

[0032] As discussed above, the particles of the present invention arecharacterized by the presence of at least two segments. A segmentrepresents a region of the particle that is distinguishable, by anymeans, from adjacent regions of the particle. Segments of the particlebisect the length of the particle to form regions that have the samecross-section (generally) and width as the whole particle, whilerepresenting a portion of the length of the whole particle. In preferredembodiments of the invention, a segment is composed of differentmaterials from its adjacent segments. However, not every segment needsto be distinguishable from all other segments of the particle. Forexample, a particle could be composed of 2 types of segments, e.g., goldand platinum, while having 10 or even 20 different segments, simply byalternating segments of gold and platinum. A particle of the presentinvention contains at least two segments, and as many as 50. Theparticles of the invention preferably have from 2-30 segments and mostpreferably from 3-20 segments. The particles may have from 2-10different types of segments, preferably 2 to 5 different types ofsegments.

[0033] A segment of the particle of the present invention is defined byits being distinguishable from adjacent segments of the particle. Theability to distinguish between segments includes distinguishing by anyphysical or chemical means of interrogation, including but not limitedto electromagnetic, magnetic, optical, spectrometric, spectroscopic andmechanical. In certain preferred embodiments of the invention, themethod of interrogating between segments is optical (reflectivity).

[0034] Adjacent segments may even be of the same material, as long asthey are distinguishable by some means. For example, different phases ofthe same elemental material, or enantiomers of organic polymer materialscan make up adjacent segments. In addition, a rod comprised of a singlematerial could be considered to fall within the scope of the-inventionif segments could be distinguished from others, for example, byfunctionalization on the surface, or having varying diameters. Alsoparticles comprising organic polymer materials could have segmentsdefined by the inclusion of dyes that would change the relative opticalproperties of the segments.

[0035] The composition of the particles of the present invention is bestdefined by describing the compositions of the segments that make up theparticles. A particle may contain segments with extremely differentcompositions. For example, a single particle could be comprised of onesegment that is a metal, and a segment that is an organic polymermaterial.

[0036] The segments of the present invention may be comprised of anymaterial. In preferred embodiments of the present invention, thesegments comprise a metal (e.g., silver, gold, copper, nickel,palladium, platinum, cobalt, rhodium, iridium); any metal chalcognide; ametal oxide (e.g., cupric oxide, titanium dioxide); a metal sulfide; ametal selenide; a metal telluride; a metal alloy; a metal nitride; ametal phosphide; a metal antimonide; a semiconductor; a semi-metal. Asegment may also be comprised of an organic mono- or bilayer such as amolecular film. For example, monolayers of organic molecules or selfassembled, controlled layers of molecules can be associated with avariety of metal surfaces.

[0037] A segment may be comprised of any organic compound or material,or inorganic compound or material or organic polymeric materials,including the large body of mono and copolymers known to those skilledin the art. Biological polymers, such as peptides, oligonucleotides andpolysaccharides may also be the major components of a segment. Segmentsmay be comprised of particulate materials, e.g., metals, metal oxide ororganic particulate materials; or composite materials, e.g., metal inpolyacrylamide, dye in polymeric material, porous metals. The segmentsof the particles of the present invention may be comprised of polymericmaterials, crystalline or non-crystalline materials, amorphous materialsor glasses.

[0038] Segments may be defined by notches on the surface of theparticle, or by the presence of dents, divits, holes, vesicles, bubbles,pores or tunnels that may or may not contact the surface of theparticle. Segments may also be defined by a discemable change in theangle, shape, or density of such physical attributes or in the contourof the surface. In embodiments of the invention where the particle iscoated, for example with a polymer or glass, the segment may consist ofa void between other materials.

[0039] The length of each segment may be from 10 nm to 50 μm. Inpreferred embodiments the length of each segment is 50 nm to 20 μm. Theinterface between segments, in certain embodiments, need not beperpendicular to the length of the particle or a smooth line oftransition. In addition, in certain embodiments the composition of onesegment may be blended into the composition of the adjacent segment. Forexample, between segments of gold and platinum, there may be a 5 nm to 5μm region that is comprised of both gold and platinum. This type oftransition is acceptable so long as the segments are distinguishable.For any given particle the segments may be of any length relative to thelength of the segments of the rest of the particle.

[0040] As described above, the particles of the present invention canhave any cross-sectional shape. In preferred embodiments, the particlesare generally straight along the lengthwise axis. However, in certainembodiments the particles may be curved or helical. The ends of theparticles of the present invention may be flat, convex or concave. Inaddition, the ends may be spiked or pencil tipped. Sharp-tippedembodiments of the invention may be preferred when the particles areused in Raman spectroscopy applications or others in which energy fieldeffects are important. The ends of any given particle may be the same ordifferent. Similarly, the contour of the particle may be advantageouslyselected to contribute to the sensitivity or specificity of the assays(e.g., an undulating contour will be expected to enhance “quenching” offluorophores located in the troughs).

[0041] In many embodiments of the invention, an assembly or collectionof particles is prepared. In certain embodiments, the members of theassembly are identical, while in other embodiments, the assembly iscomprised of a plurality of different types of particles. In embodimentsof the invention comprising assemblies of identical particles, thelength of substantially all of the particles for particles in the 1 μm-15 μm range may vary up to 50%. Segments of 10 nm in length will vary±5 nm while segments in 1 μm range may vary up to 50%. The width ofsubstantially all of the particles may vary between 10 and 100%preferably less than 50% and most preferably less than 10%.

[0042] The present invention includes assemblies or collections ofnanobar codes made up of a plurality of particles that aredifferentiable from each other. Assembly or collection, as used herein,does not mean that the nanoparticles that make up such an assembly orcollection are ordered or organized in any particular manner. Such anassembly is considered to be made up of a plurality of different typesor “flavors” of particles. In some such assemblies, each of the nanobarcodes of the assembly may be functionalized in some manner. In manyapplications, the functionalization is different and specific to thespecific flavor of nanoparticle. The assemblies of the present inventioncan include from 2 to 10¹² different and identifiable nanoparticles.Preferred assemblies include more than 10, more than 100, more than1,000 and, in some cases, more than 10,000 different flavors ofnanoparticles. The particles that make up the assemblies or collectionsof the present invention are segmented in most embodiments. However, incertain embodiments of the invention the particles of an assembly ofparticles do not necessarily contain a plurality of segments.

[0043] In certain embodiments of the invention, the particles of thepresent invention may include mono-molecular layers. Such mono-molecularlayers may be found at the tips or ends of the particle, or betweensegments. Examples of the use of mono-molecular layers between segmentsare described in the section entitled ELECTRONIC DEVICES in U.S. Utilityapplication Ser. No. 09/598,395, filed Jun. 20, 2000.

[0044] The present invention is directed to the manufacture offreestanding nanobar codes. By “freestanding” it is meant that nanobarcodes that are produced by some form of deposition or growth within atemplate have been released from the template. Such nanobar codes aretypically freely dispensable in a liquid and not permanently associatedwith a stationary phase. Nanobar codes that are not produced by someform of deposition or growth within a template (e.g., self-assemblednanobar codes) may be considered freestanding even though they have notbeen released from a template. The term “free standing” does not implythat such nanoparticles must be in solution (although they may be) orthat the nanobar codes can not be bound to, incorporated in, or a partof a macro structure. Indeed, certain embodiments of the invention, thenanoparticles may be dispersed in a solution, e.g., paint, orincorporated within a polymeric composition.

[0045] The particles of the present invention may be prepared by avariety of processes. The preferred process for the manufacture of aparticular particle can often be a function of the nature of thesegments comprising the particle. In most embodiments of the invention,a template or mold is utilized into which the materials that constitutethe various segments are introduced. Defined-pore materials are thepreferred templates for many of the preferred particles of the presentinvention. Al₂O₃ membranes containing consistently sized pores are amongthe preferred templates, while photolithographically prepared templates,porous polycarbonate membranes, zeolites and block co-polymers may alsobe used. Methods for forming segments of particles includeelectrodeposition, chemical deposition, evaporation, chemical selfassembly, solid phase manufacturing techniques and photolithographytechniques. Chemical self assembly is a method of forming particles frompreformed segments whereby the segments are derivatized and a chemicalreaction between species on different segments create a juncture betweensegments. Chemically self-assembled nanoparticles have the uniqueability of being controllably separated between segments by reversingthe chemical bond formation process.

[0046] One of the preferred synthetic protocols used to prepare metallicnanobar codes according to the embodiments of the present invention isan extension of the work of Al-Mawlawi et al. (Al-Mawlawi, D.; Liu, C.Z.; Moskovits, M. J. Mater. Res. 1994, 9, 1014; Martin, C. R. Chem.Mater. 1996, 8, 1739) on template-directed electrochemical synthesis.See, Example 1, below. In this approach, metals are depositedelectrochemically inside a porous membrane. The synthetic method of thepresent invention differs from previous work in several respectsincluding the following. First, the electroplating is done withagitation, such as in an ultrasonication bath. Second, the temperatureis controlled, for example, by using a recirculating temperature bath.These first two modifications increase the reproducibility andmonodispersity of rod samples by facilitating the mass transport of ionsand gases through the pores of the membrane. Third, rods with multiplestripes are prepared by sequential electrochemical reduction of metalions (e.g., Pt²⁺, Au⁺) within the pores of the membranes. Because thelength of the segments can be adjusted by controlling the amount ofcurrent passed in each electroplating step, the rod resembles a “barcode” on the nanometer scale, with each segment length (and identity)programmable in advance. While the width of the rods and the segmentlengths are generally of nanometer dimensions, the overall length isgenerally such that it can be visualized directly in an opticalmicroscope, exploiting the differential reflectivity of the metalcomponents.

[0047] There are many parameters in the nanorod synthesis that aretunable, such that it is theoretically possible to generate manymillions of different patterns, uniquely identifiable by usingconventional optical microscopy or other methods. The most importantcharacteristic that can be changed is the composition of the stripedrods. The simplest form of a nanoparticle is one with only one segment.To this end, several different types of these solid bar codes have beenprepared. By simply using only one plating solution during thepreparation, a solid nanoparticle is produced.

[0048] To generate two-segment nanobar codes, two metals (e.g., Au, Ag,Pd, Cu, etc.) can be electroplated sequentially, or simultaneously toform alloys. Nanobar codes can also be generated using 3 differentmetals. Synthesis of a Au/Pt/Au rod may be accomplished with 1 C of Au,8 C Pt, and 1 C of Au. The nominal dimensions of the segments are 1 μmof Au, 3 μm of Pt, 1 μm of Au. The 5-segment nanobar codes,Ag/Au/Ag/Au/Ag, were generated by sequentially plating the appropriatemetal. In some embodiments it is possible to include all metals insolution but control deposition by varying the charge potential current.A nine-segment nanobar code, Au/Ag/Au/Ag/Au/Ag/Au/Ag/Au has also beenprepared. The number of segments can be altered to desiredspecifications.

[0049] The next controllable factor is diameter (sometimes referred toherein as width) of the individual rods. Many of the nanobar codesdescribed were synthesized using membranes with a pore diameter of 200nm. By altering the pore diameter, rods of differing diameter can bemade. Au rods have been synthesized in a membrane that has 10 nmdiameter pores, 40 nm pores and pores in the range of 200-300 nm.

[0050] The ends of the rods typically have rounded ends or flat ends. ATEM image of an Au rod that was made by reversing the current flow (fromreduction at −0.55 mA/cm² to oxidation at +0.55 mA/cm²) and removingsome of the gold from the tip of the rod generated a spike extendingfrom the tip of the rod. Additionally, branched ends can be generated.This can be typically controlled by controlling the amount of metal thatis plated into the membrane. The edges of the membrane pores have atendency to be branched which lead to this type of structure.

[0051] An additional way to alter the ends of the rods is to control therate of deposition. Gold rods (2 C total, 3 μm) were plated at a currentdensity of 0.55 mA/cm². Then the current density was reduced to 0.055mA/cm² and 0.1 C of Au was plated. The last segment of gold deposits isa hollow tube along the walls of the membrane.

[0052] Example 1 describes the manufacture of single flavors ofnanoparticles according to one embodiment of the invention.

[0053] In order to produce many thousands of flavors of nanorods, inpractical quantities, and to attach molecules to most or all, novelcombinatorial or multiplexed synthesis techniques are necessary. Severalsynthesis embodiments are included within the scope of the invention.Each approach has advantages and disadvantages depending on the specificapplication and the required number of types and total number ofnanorods needed for the application.

[0054] The present invention includes methods of manufacture ofnanoparticles that allow for the simultaneous or parallel manufacture ofa plurality of different flavors of nanobar codes.

[0055] Prior to the present invention, no system or apparatus has beendescribed whereby it was possible to prepare more than one type ofnanobar code simultaneously or in parallel. In the preferred embodimentsof this invention, such method for the simultaneous manufacture ofnanobar codes allows for the manufacture of 2 or more, more than 5, morethan the 10 and preferably more than 25 different flavors of nanobarcodes. By simultaneous or parallel it is meant that common elements areemployed in the manufacture of the more than one nanobar code. Forexample, in the apparatus depicted in FIGS. 1 and 2, there are 25separate membranes, each with a separately controllable electrodeconnection on the back side, but with common access to the platingsolution. In other embodiments, the separate membranes (or regions on asingle membrane) may have a common electrode, but separatelycontrollable solution access. In still other embodiments, thesimultaneous manufacture of different types of nanoparticles is commonlycontrolled. Any system or apparatus whereby a plurality of differentflavors of nanoparticles (e.g, particles having a plurality of segments,that are 10 nm to 50 μm in length, and have a width from 5 nm to 50 μmthat are differentiable from each other) can be prepared in parallel isincluded within the scope of this invention. Among the options that canbe employed to effect this parallel manufacture are the following:

[0056] 1. Multi-electrode and Microfluidic Synthesis: To synthesize manyflavors of nanorods on a single membrane, the membrane can be dividedinto separate electrical zones, with each zone using a different platingrecipe. Of course, several smaller membranes could be used, one for eachseparate zone, as opposed to a single membrane with multiple zones. Theelectrical zone approach can be achieved by patterning the Agevaporation that initially seals one side of the membrane into manyseparate islands. Each island would have its own electrode, and controlcircuitry can activate each island separately for plating. Themicrofluidic approach utilizes a single evaporated Ag electrode, butwould divide the opposite side of the membrane into separate fluidicregions, and control the flow of plating solutions to each region. Bothof these techniques may be automated, and result in the synthesis ofhundreds of nanorod flavors per membrane. Thousands to millions offlavors is probably not practical with either of these approaches due topractical limitations in the number of electrical or fluidic connectionsto the membrane

[0057] 2. Patterned front-side insulation: This approach appliesinsulating patterned coatings (e.g., photoresist) to the front-side(electrodeposition side) of a membrane. Where the membrane is coated,electroplating is inhibited. The coating can be removed and reappliedwith different pattern between electroplating steps to achieve synthesisof many flavors of nanobarcode within one membrane.

[0058] 3. Patterned back-side insulation: This approach appliesinsulating patterned coatings (e.g., photoresist) to the back-side(electrode side) of a membrane, which is divided into many separateelectrical contacts. Where the electrode is coated, electroplating isinhibited. The coating can be removed and reapplied with differentpatterns between electroplating steps to achieve synthesis of manyflavors of nanobarcode within one membrane.

[0059] 4. Lithography vertical or horizontal: This technique, thatoffers increased design flexibility in the size and shape of nanorods,utilizes lithographic processes to pattern the deposition of multiplelayers of metals on a silicon substrate. This approach takes advantageof the tremendous capabilities developed in microelectronics and MEMS,and promises very high quality nanorods with greater design flexibilityin the size and shape of nanorods than membrane-based techniques. Eachof these synthetic approaches must be mated to complementary well arraysto allow nanobar release into separate vessels.

[0060] 5. Light-addressable electroplating: A further technique thatcould produce thousands of flavors in one synthesis step also utilizesmembrane-based synthesis, but includes light-directed control of theelectroplating process. In this technique, a light-addressablesemiconductor device is used to spatially modify the electricalpotentials in the vicinity of the membrane, and thus spatially modulateelectroplating currents. In this manner, the membrane is opticallysubdivided into many different zones, each of which produces a differentflavor of nanorod.

[0061] 6. Electrical multiplexing to multiple separate templatemembranes immersed in common plating solution: In this approach,multiple template membranes are immersed in a common plating solution,with a common anode electrode (platinum). Each membrane has a separateelectrical connection from a computer-controlled current and/or voltagesource to its silver-coated backside.

[0062] 7. Template Dicing: A template may be cut into a number ofsmaller pieces. This may be accomplished, for example, using a dicingsaw or by a “scribe and break” procedure where the wafer is cut part ofthe way through and then broken; the latter may be preferred in someembodiments because it generates less dust and debris.

[0063] Several of these embodiments are based on existing proceduresusing defined-pore membranes. (i) One technique generates hundreds toperhaps a few thousand types of nanorods, by lithographically patterningthe backside silver that is deposited on the membrane into isolatedislands, each island forming an individually addressable electricalcontact. By way of example, each island would have enough surface areato contain between 10⁶ and 10⁸ individual rods, all of the same type.(Note that since the membrane thickness, and therefore pore length, ismuch greater than the nanorod length, multiple nanorods can besynthesized in each pore. Each nanorod may be separated from others inthe same pore by a silver plug that would later be dissolved. This couldincrease the total yield by 10×.) The membrane is then placed, withcareful registration, onto a “bed-of-nails” apparatus, with individualspring-loaded pins contacting each electrode on the membrane.Computer-controlled circuitry attached to the bed-of-nails is able toindividually turn on or off each electrode. During the electroplatingprocess, each island would be plated with unique combinations of metaltypes and thicknesses. In this manner, each island would produce rods ofdifferent lengths, different numbers of stripes, and different materialcombinations, allowing ultimate design flexibility. (ii) The aboveapproach will be limited in the number of types of rods that can besynthesized by the reliability and packing density of the bed-of-nailsapparatus. To avoid this limitation, the bed-of-nails apparatus can bereplaced by a liquid metal contact. To prevent the liquid bath fromsimultaneously contacting every electrode, the backside of the membranemay be patterned with a nonconductive coating. To individually addresselectrodes during synthesis, the pattern would be removed and replacedwith a different pattern between electroplating steps. This approachwill enable a much higher density of isolated islands, and thereforemore types of rods to be synthesized. With island spacing of 100microns, which would be trivial to achieve using lithographicalpatterning, up to 10⁵ types of rods could be synthesized. Since thetotal number of pores in each membrane is a constant there will beproportionally fewer rods of each type. (iii) The above two approachesuse commercially available aluminum oxide membrane filters, which havepore size and density that are suitable for nanorod synthesis. However,the membrane thickness is typically greater than that required, whichcan cause variability in rod and stripe lengths due to non-uniform masstransport into the pores during electroplating. Also, the largest poresavailable in these membranes (and thus nanorod widths) are 250 nm, andit would be desirable for some applications to have rod widths of 1micron or more (this could also be used for embodiments with widths ofless than 1 μm).

[0064] To address these issues, pore matrices may be constructed usingphotolithography techniques, which will give ultimate control over thepore dimensions and lengths, and increase the design flexibility andquality of the resulting nanorods. According to this embodiment apositive photoresist-coated substrate is exposed to an interferencepattern of light, using a technique similar to that used forinterference-lithography generated diffraction gratings. Typically, thesubstrate is a silicon wafer, with (a) a thin coating of a conductivematerial, such as titanium nitride, or gold, (b) a thick coating ofpolymer, such as polymethylmethacrylate (PMMA) or polyimide, (c) an etchstop, such as SiO₂, aluminum, or nickel, and (d) a photoresist. Exposureand subsequent development yields a two-dimensional array of pores inthe photoresist. Reactive ion etching may then be used to transfer thepore pattern down through the polymer layer. The photoresist layer isremoved, and the conductive layer under the polymer becomes the cathodefor electroplating into the pores. The shape and diameter of thenanorods can be controlled by the mask or by adjusting the light sourceand the resultant standing wave pattern. For most applications, aconventional mask is preferred. However, interference lithographytechniques may be preferred when the desired pore diameter is lower thanthe resolution limit available from state of the art projectionlithography tools. Achieving smaller pore size may also benefit from theuse of x-ray or e-beam etching.

[0065] An advantage to this technique is that the template thickness,which may be the same as pore length, can be tailored to the length ofthe rods, which may improve uniformity of electroplating across themembrane. With this technique, 10¹⁰ to 10¹² nanorods can be constructedon a single substrate. The two approaches described above can beutilized to synthesize many types of nanobar code from a single wafer.(iv) A further approach uses the customized lithographically-definedpores from above, and achieves the ultimate in design flexibility byusing novel light-directed electroplating. The template pores areconstructed just as in the third approach, but on top of aphotosensitive semiconductor wafer. The pore-side of the wafer isimmersed in an electroplating reagent, and the other side is illuminatedwith patterns of light. Light exposure is used to generate photocurrentin the wafer, and switch the plating current on or off for eachconductive zone within the wafer. A computer-controlled spatial lightmodulator selectively illuminates different zones at different times, sothat each zone will be subjected to a different computer-controlledplating recipe. Depending on the resolution of the optical system thatexposes the wafer, this could result in 10⁴ to 10⁶ separate flavors ofnanorods synthesized on a single wafer. With 10¹² total pores per wafer,10⁶ to 10⁸ nanorods of each flavor could be synthesized.

[0066] Membranes with extremely high densities of uniform pores can becreated by photolithographic techniques and the resulting nanoparticlesare of very uniform size and length. Using the methods described herein,a 4-inch silicon wafer can serve as the substrate for the formation of,for example, 50 billion pores of diameter 200 nm and period 400 nm.Pores of smaller diameter and lower period are readily achievable.Unlike the pores formed in anodized alumina membranes or polycarbonatetrach-etched membranes, pores formed by photolithographic techniqueshave a very tight diameter distribution, do not overlap or branch, andare straight and parallel.

[0067] Interference Lithography and Achromatic InterferenceLithography-Based Methods for Nanoparticle Template Formation

[0068] In one series of embodiments, membrane templates are formed fromresist-coated substrates or multi-layer stacks by means of interferencelithography (also known as “holographic” or “interferometric”lithography). Interference lithography (IL) is well known in thesemiconductor and microfabrication arts as a technique capable ofpatterning grids and gratings over a large area of resist (up to 10 cmdiameter) without using a mask. Briefly, IL involves forming an opticalstanding wave through the intersection of two laser beams. The standingwave creates a line of alternating exposed and unexposed regions on theresist. By exposing a resist first in one orientation, and then at 90°to the first orientation, a “grid” is patterned on the resist. Thispattern can be transferred into material that lies underneath theresist, thereby forming pores, by developing the resist and thenperforming an etch. The resist and/or the underlying material form thewalls of the membrane pores within which nanoparticle can subsequentlybe formed.

[0069] The period of the patterns that interference lithography canexpose is given by the equation p=λ/(2 sin θ), wherein λ is thewavelength, and θ is the half angle between the intersecting beams.Interference lithography is best performed with wavelengths longer than248 nm; this corresponds to a period of approximately 200 nm. Togenerate patterns of lower period, shorter wavelength laser light isrequired. However, lasers that provide shorter wavelength lightgenerally do not produce sufficiently monochromatic light to generaterobust interference patterns.

[0070] To form patterns with a period less than 200 nm, achromaticinterference lithography (AIL) is preferred. An optical system forgenerating 100 nm period patterns with 193 nm light from a ArF excimerlaser, for example, is described in Savas et al., J. Vac. Sci. Tech.1996, 14, 4167, incorporated herein by reference in its entirety.Briefly, achromatic interference lithography uses a first phase gratingto generate two first-order light beams from an incident light source.Two further phase gratings recombine these divergent light beams bysecond-order diffraction. In the case of 193 nm light from the ArFlaser, each phase grating has a period of 200 nm and each is fabricatedby interference lithography and reactive ion etching. The resultinglight beam takes the form of a standing optical wave of 100 nm periodwhich can extend over a 10 cm diameter area. Orthogonal exposure of aresist-coated substrate to the standing optical wave can form an arrayof pores in the same way as described above for interferencelithography.

[0071] In a typical embodiment, EL or AIL is performed on multi-layerstack, comprising a substrate on which has been deposited a conductivematerial layer, a polymer layer, an etch stop layer, and a photoresist.Following exposure by the orthogonal standing optical waves, the resistcan be developed by techniques well known in the art. The grid patternin the developed resist can then be transferred down into the etch masklayer, and the polymer layer by any of the etching techniques known inthe art, including wet etching, dry etching, reactive ion etching,electron beam and laser writing; reactive ion etching is preferred. Inthis configuration, each pore passes through the resist layer, the etchmask layer, and the polymer layer. Optionally, the resist layer can becompletely removed following etching so that the pores are formed solelywithin the polymer layer. The depth of the pores, and hence the lengthof nanoparticles that can be formed in the pores, is determined by thethickness of the relevant layers of the stack.

[0072] The conductive material may be a metal, a metal-containingcompound, or a metal alloy, including without limitation titaniumnitride, nickel, copper, zinc, silver and gold; titanium nitride andgold are preferred. The conductive layer may be deposited by anysuitable means, including sputtering. To promote adhesion between theconductive layer and the substrate, a layer of adhesion promotingmaterial may be deposited on the substrate before deposition of theconductive layer. Adhesion promoting materials include titanium andcromium; titanium is preferred where titanium nitride is the conductivematerial; cromium is preferred where gold is the conductive material.

[0073] The polymer may be polyimide, polymethylmethacrylate (PMMA),photoresist, or other suitable polymer known in the art. Althoughreferred to herein as the “polymer” layer, to the extent it may beconsidered as limiting the scope of the invention, that label should beignored. Thus, the “polymer” layer includes materials that can be etchedaccording to the methods of the present invention to form pores even ifthose materials are not polymeric (e.g., polysilicon, SiO₂). The etchstop layer may be SiO₂, aluminum, and nickel, or other etch stop know inthe art. Suitable resists include both positive and negativephotoresists known in the art. Positive photoresists are preferred forfeatures less than 3 μm in size. It will be understood by those skilledin the art that there are many suitable resists (including positive andnegative resists), etch stops and polymers that can be patterned intogrids using IL and AIL. Any stack that can be patterned by IL or by AILto yield pores suitable for the formation of nanoparticles iscontemplated by the present invention. Similarly, although siliconwafers are the preferred substrates of the invention, many othersuitable substrates are known in the art.

[0074] An antireflective coating (ARC) may be included in the stack.Such a coating acts to prevent the reflection of light by the underlyingsubstrate, thereby assisting the integrity of the standing optical wave.

[0075] In embodiments where high etch rate selectivity is not desired,the etch stop layer may be omitted from the stack. The conductive layermay also be omitted if the substrate is sufficiently conductive (e.g.,if it has been doped) or if the nanoparticles are to be made by aprocess other than electrodeposition (e.g., CVD), and therefore do notrequire an electrode. Indeed, in the most basic embodiments of theinvention, even the polymer layer may be omitted, so that thephotoresist will form the walls of the pores.

[0076] Mask-Based Photolithographic Methods for Nanoparticle TemplatesFormation

[0077] Membrane templates for the formation of nanoparticles also may beformed by conventional mask-based photolithography techniques well-knownin the semiconductor and microfabrication arts. In these embodiments,masks with a grid pattern generated by standard methods known in the art(e.g., e-beam writing and laser writing) are used to expose aresist-coated substrates and multi-layer stacks. The substrates andmulti-layer stacks, as well as the techniques for development andetching thereof, are preferably based on those described above (e.g., asubstrate overlaid first with a polymer layer, an etch stop material,and a photoresist).

[0078] An advantage of using masks is that each mask can be used anumber of times, thereby obviating the need to use the AIL or IL opticalconfiguration every time a membrane template is required. The use of anytype of mask known in the art is contemplated by the invention. In someembodiments of the mask-based approach, the mask itself may be formed byIL or AIL.

[0079] If the mask is a grating, then two orthogonal exposures of theresist are necessary to pattern the resist with a grid; if the mask is agrid, then a single resist exposure can be used to pattern a grid.Methods for forming free-standing grating and grid masks by IL and AILare described in, for example, Wolf and Tauber, Silicon Processing forthe VLSI Era, Vol. 1 Process Technology (2nd Ed.) Lattice Press,California (2000), incorporated herein by reference in its entirety.Once a mask has been formed, it can be used to expose a resist by, forexample, contact or proximity exposure techniques known in the art.

[0080] Use of Membrane Templates

[0081] The membrane templates produced by the methods of the presentinvention, whether through IL, AIL, or mask-based photolithography, canbe used to form nanoparticles via a number of techniques. Mostpreferably, electrochemical deposition is used, requiring that themembrane template have a conductive material in communication with thepores. This may be achieved, for example, by depositing a layer ofconductive material overlying the substrate, as described above, dopingthe substrate so that it can act as the electrode, or both.

[0082] In other embodiments, material can be deposited within pores bychemical vapor deposition (e.g., organic-metallic vapor deposition) orevaporation. For example, electron-beam evaporation has been used todeposit metal within pores formed by AIL. Savas et al., J. AppliedPhysics 1999, 85, 6160, incorporated herein by reference in itsentirety. Methods for the formation of nanoparticles in membranetemplate pores by evaporative techniques are discussed further herein.

[0083] In the aforementioned embodiments in which the nanoparticles areformed by electrodeposition into pores of membrane template, there mustbe a conductive material in communication with the pore so that currentmay be applied. In some embodiments, the substrate may first be coatedwith one or more conductive layers before the stack is built. Thephotoresist is then exposed and developed, as described above. When thestack is etched down to the conductive layer, the resulting porescomprise a layer of conductive material at their base. By applying acurrent to the substrate, the conductive layer acts as an electrode atthe base of each pore, thereby allowing charged material to beelectrodeposited within the pore. In this way, segmented nanoparticlescan be built through sequential electrochemical deposition according tothe methods provided herein. Other configurations allowing conductivematerial to be in communication with the pore are possible (e.g.,additional conductive material may be deposited within the pore, thewalls of the pore may comprise conductive material, there may beconductive pillars or posts within the pore, and so on.). As discussedabove, in preferred embodiments, the conductive layer comprises a layerof Ti overlaid with a layer of TiN, or Cr overlaid with a layer of Au.Preferably the Ti or Cr layer is about 5 nm thick and the TiN or Aulayer is about 20 nm thick.

[0084] When the electrochemical deposition procedure is complete, thematerial that forms the walls of the pore (e.g., PMMA) can be removed,exposing the nanoparticles. The nanoparticles can be released from thesilicon wafer by a number of methods. For example, the nanoparticles canbe liberated by physically breaking the linkage between the wafer andthe nanoparticles using, for example, sonication or high-pressure water.The nanoparticles also may be liberated by etching the silicon wafer,for example using HF acid. All of the aforementioned techniques forliberating nanoparticles are equally applicable in embodiments wherenanoparticles are formed in pores through techniques other thanelectrochemical deposition.

[0085] Alternatively, the nanoparticles may be released by dissolvingthe conductive layer. Indeed, areas of sacrificial conductive materialmay be deposited on the conductive layer such that they lie at thebottom of the pores. Because it is conductive, the material can transmitthe current to the growing nanoparticles. Selected so that it is easilydissolved or otherwise removed, the sacrificial conductive material maybe useful in liberating the nanoparticles formed in the pores. A numberof materials may be used as sacrificial conductive materials, includingwithout limitation Ag, Cu, and Zn. In embodiments in which Ag is used asthe sacrifical conductive material, nitric acid can be used to liberatethe nanoparticles; when Cu is the sacrifical conductive material,sulfuric acid can be used; when Zn is the sacrifical conductivematerial, a weak acid can be used. It should be noted that the use of aparticular metal as the sacrificial conductive layer will preclude theuse of that metal as one of the segments of the nanoparticle.

[0086]FIG. 3 illustrates schematically an embodiment of the inventionusing a four-layer stack on a silicon wafer substrate 101. In FIG. 3A,the stack is shown before exposure to radiation. Substrate 101 isoverlaid with conductive layer 102, then with polymer layer 103, thenwith a etch stop layer 104, and finally with photoresist layer 105. InFIG. 3B, the stack is shown following exposure and development of thephotoresist, followed by etching down to etch stop 104, thereby formingpores 106. Exposure may be performed using IL or AlL (with twoorthogonal exposures), or using conventional mask-based photolithographywith a grid mask. In FIG. 3C, further etching down through polymer layer103 to conductive layer 102 results in the formation of pores 107. InFIG. 1D, nanoparticles 108 are formed within pores 107 byelectrochemical deposition using the conductive layer 102 as the platingelectrode. Finally, in FIG. 1E, free-standing nanoparticles 109 areliberated by dissolving the conductive layer 102.

[0087] While a single synthesis process using one silicon wafer canyield tens of billions of identical nanobarcodes, it may be desirable tomanufacture many different codes simultaneously on a single wafer toprovide for the diversity of nanobarcodes required for manyapplications. This can be achieved with a straightforward modificationto the processes described above, by patterning the conductive layerimmediately below the resist stack into separate zones. Each zone canthen be connected to a separate electrical control system.

[0088] Reuseable Membrane Templates

[0089] In still further embodiments, the membrane template created usingphotolithographic techniques is re-usable. This can be achieved in thefollowing way. Step 1: a layer of polysilicon is deposited on a siliconwafer using either CVD or PECVD deposition. Step 2: A layer ofphotoresist is spun on the silicon wafer. Step 3: The resist is exposedusing a mask, IL, or AIL, to pattern a grid. Step 4: The resist isdeveloped and the polysilicon is etched, revealing an array of pores inthe polysilicon. Step 5: A sacrificial layer of conductive metal isdeposited, electrochemically or by CVD, inside the pores that wereetched into the polysilicon. For example, Zn can be used as it is easilydissolved in a weak acid. Step 6: A layer of silicon dioxide is thendeposited on the surface of the stack, followed by a hydrogen bake toremove metal oxide formed in the silicon deposition process. Step 7:Nanoparticles may be formed inside the pores using CVD orelectrochemical deposition; in the latter case, the Zn may participateas an electrode. Step 8: the silicon dioxide deposited in step 7 isdissolved using HF. Step 9: The Zn deposited in step 6 is dissolved,thereby liberating the nanoparticles. The membrane template can bere-used by returning to step 5.

[0090] Photolithographic Formation of Nanoparticles By Etching aPre-formed Film Stack

[0091] The aforementioned embodiments are all directed to the formationnanoparticles in porous membrane templates made by photolithographictechniques. In another series of embodiments, photolithographictechniques are used to etch nanoparticles from a pre-formed stack ofmaterial, wherein each layer of the stack corresponds to a particularsegment of the subsequent nanoparticle. In one such embodiment of theinvention, layers of material, such as metal films, are deposited onto asilicon wafer to form a stack. A layer of photoresist is then spun onthe material stack. The stack is then exposed to radiation (e.g., UVlight) by conventional mask-based photolithography (or by IL or by AIL)to pattern a grid on the resist. Following development of the resist,the entire film stack is then etched revealing many cylindrical filmstacks. The nanoparticles can be liberated by one of the methodsdescribed above (e.g., physically, or by dissolving a sacrificial baselayer).

[0092] Alternatively, photolithography techniques may be used tosynthesize the nanoparticles one layer at a time. Thus, in a furtherembodiment of the invention, a layer of material is depositied on asilicon wafer, via electrochemical deposition or CVD, to form Film 1(corresponding to the first segment of the nanoparticle). Photoresist isthen spun on top of Film 1. The resist is exposed, e.g., using a mask orby IL or AIL, to pattern an array of cylindrical posts (i.e., theopposite resist pattern to a pore pattern). Following resistdevelopment, the film is etched with an appropriate wet or dry etchmethod to leave cyclindrical posts of the Film 1 material. To form thesubsequent segments of the nanoparticle, the preceding steps arerepeated n times, depositing and etching Films 2 through n, to form thesegments in the nanoparticle. The nanoparticles can be liberated by oneof the methods described above.

[0093] The use of photolithographic techniques provides an opportunityto make rods from many different materials beyond simple metals. Forexample, glass can be spin-coated on a planar substrate and readilyetched later. Glass in each layer could be doped with metals orphosphorescent materials so that each layer would fluoresce a differentamount. When the striped rods are liberated, the codes would be readwith a fluorescent imaging system. Metals and metal oxides can bedeposited on a planar surface and later etched. Alternating layers ofmetals and oxides can provide high contrast segments that can easily beread by a reflectance microscopy. It should be noted that there arenumerous other materials that can be used to prepare membranes ortemplates for nanorod synthesis. One example of many are bundles ofoptical fibers in which the cores are etchable under conditions wherethe claddings are not. Carrying out this etching, followed by slicingacross the bundle, yields a membrane with hole diameters the size of thefiber cores. Note that fibers can be drawn out (using heat) to submicrondiameters. Note also that fiber bundles with collections of greater than1,000,000 fibers are commercially available; this could easily beextended to 10 million. Another group of materials that could be used,for example, are molecular sieve materials with well-defined cavitiessuch as zeolites.

[0094] Note also that other methods can be used to prepare templates ormembranes from a variety of different methods. Such methods include butare not limited to: MEMS, electron beam lithography, x-ray lithography,uv-lithography, deep lithography, projection lithography, standing wavelithography, interference lithography, and microcontact printing.

[0095] Chemical self-assembly/deassembly methods may also be used. Forexample, formation of an infinite, close-packed, 2-dimensional hexagonallayer of latex balls on a planar surface has been demonstrated. Suchparticles could be shrunk by 10% in size, e.g., by cooling thetemperature. Then a polymer may be grown in the spaces between theinfinite 2-D array (that is no longer close packed). Then the balls areselectively dissolved, leaving behind a polymeric material with welldefined holes equal to the final diameter of the latex balls.

[0096] The particles of the present invention may also be prepared inlarge scale by automating the basic electroplating process that isdescribed in Example 1. For example, an apparatus containing a series ofmembranes and separate electrodes can be used to make a large number ofdifferent flavors of nanoparticles in an efficient computer controlledmanner. An example of this type of apparatus is depicted in FIGS. 1 and2.

[0097] The embodiment of the invention depicted in FIGS. 1 and 2synthesizes 25 types of nanobar codes simultaneously in 25 separatetemplate membranes (e.g., Whatman Anodisc membranes, 25 mm diameter, 60micron thick, with 200 nm pores) mounted in a liquid flow cell. Beforemounting the membranes in the flow cell, each membrane is silver-coatedon one side (which is the branched-pore side of the membrane) in avacuum evaporator. Then each membrane is immersed in a silver platingsolution with electrodes on both sides, and additional silver iselectroplated onto the evaporated silver coating and into the pores (at4 mA for about 30 minutes), to completely close all of the membranepores. Each membrane is then mounted with its silver-coated side incontact with an electrode in the flow cell. The flow cell is about 1.5mm thick, containing about 30 ml of liquid. Opposite the membranes is aplatinum mesh electrode with surface area slightly larger than theentire 5×5 array of membranes.

[0098] The flow cell can be filled (by computer control) with water,nitrogen gas, gold plating solution (e.g., Technics), silver platingsolution (e.g.,Technics Silver Streak and/or additional platingsolutions). The flow cell is in thermal contact with a coolant watertank, the temperature of which is controlled by recirculation through atemperature-controlled bath. In the coolant tank opposite the flow cellis an ultrasonic transducer (Crest, 250 Watt), which is turned on duringelectroplating operations to facilitate mass transport of ions and gasesthrough the membrane pores. Control software is used to automaticallyflow the appropriate solutions through the flow cell, and individuallycontrol the electroplating currents or potentials at each separatemembrane. The software also measures temperature at various locations inthe apparatus, and controls the sonicator and peristaltic pump. Thesoftware allows the user to define recipes describing the desired stripepattern for each nanobar code in the 5×5 array. The software reads therecipe, and then automatically executes all fluidic and electrical stepsto synthesize different types of nanobar codes in each membrane.

[0099] After nanorod synthesis is complete, the membranes are removedfrom the flow cell, and individually postprocessed to free the nanobarcodes from the template pores. First, each membrane is immersed inapproximately 2M HN (nitric acid) for about 30 minutes to dissolve thebackside silver coating. Then the membrane is immersed in NaOH todissolve the alumina membrane, and release the rods into solution. Therods are then allowed to settle under gravity, and the NaOH is washedout and replaced with H₂O or Ethanol for storage. In a furtherembodiment, rather than moving the solution exposed to a stationarymembrane or template, the membranes or templates may be moved from oneplating solution to another.

[0100] An apparatus for performing such manufacture of 25 types orflavors of nanobar codes is depicted in FIGS. 1 and 2. As describedabove, 25 separate membrane templates are placed in a common solutionenvironment, and deposition is controlled by the application of currentto the individual membranes. For example, membranes 1-10 may begin withthe deposition of a layer of gold that is 50 nm thick, membranes 11-20may begin with the deposition of gold that is 100 nm thick, whilemembranes 21-25 may not have an initial layer of gold. This depositionstep can be easily accomplished in the apparatus of this embodiment byfilling the solution reservoir with a gold plating solution and applyingcurrent to membranes 1-10 for the predetermined length of time,membranes 11-20 for twice as long and not at all to membranes 21-25. Thegold plating solution is then removed from the chamber and the chamberrinsed before introducing the next plating solution.

[0101] The apparatus of this embodiment has been designed to berotatable around a pivot point for ease of access to the solutionchamber and the electric and plumbing controls on the back of theapparatus. Referring to FIG. 1, the apparatus rests upon a base 101. Thepivoting mechanism is comprised of the pivoting support 103, the pivotlocking pin handle 105, and the pivot pin 107. The apparatus is equippedwith a halogen light, contained in the box 108, and a sonicator, locatedat 109, in fluid communication with a solution chamber.

[0102] The flow cell is defined by the rear cell assembly 111 and thefront cell assembly 113. The electrical connectors 115 are on the topsof the rear and front assemblies. The assemblies are held in place byclamping bolts 117 to maintain a sealed solution chamber. The 25templates 119 for nanoparticle growth are held between front and rearassemblies, and the front assembly has an electroforming cell frontwindow 121.

[0103]FIG. 2 is a cross-sectional view of the apparatus shown in FIG. 1.Many of the same elements can be seen in FIG. 2 that were defined withrespect to FIG. 1, and they have been numbered the same. FIG. 2 alsoallows visualization of cell partitioning gaskets 123 between front andrear assemblies and gasket alignment pin 125. FIG. 2 also shows rearassembly glass window 127. The water tank 129 for temperature control isfound adjacent to the rear assembly, and the halogen lamp 131 is shown.The ultrasonic apparatus is comprised of the ultrasonic transducer 133and the ultrasonic tank 135.

[0104] While the embodiment described above clearly illustrates howtwenty-five types of nanobar codes comprising cylindrical, segmentedmetal nanoparticles can be prepared by parallel synthesis, the concepthas very broad applicability. It is straightforward to extend thisembodiment to hundreds or thousands of parallel reaction chambers.Likewise, it is straightforward to extend this method to the fabricationof nanorods with three or more different materials. Likewise, it shouldbe clear that, through appropriate use of Ag spacers, that more than oneflavor of nanobar code can be prepared within a single reaction vessel.In other words, one could prepare an Au—Pt rod, deposit Ag, and thenprepare an Au—Pt—Au rod. After rod release from the membrane, Agdissolution will lead to production of two types of rods. Of course, thenumber of a single type of particles could be increased by growingmultiple copies of a single rod within the same reaction vessel.

[0105] It should likewise be realized that, rather than introduction ofone plating solution to a collection of membranes, it is straightforwardto employ microfluidics to address templates individually. In otherwords, a different plating solution could simultaneously be delivered totwo or more locations. Thus, in principle, one could be making stripesof 5 or 10 or more compositions, and with 5 or 10 or more segmentwidths, at the same time, but in different, pre-programmed locations.

[0106] Importantly, the materials chosen for this synthesis (Au, Ag, Pt)are meant to be illustrative, and in no way limiting. There are numerousmaterials that can be electrodeposited in this fashion, includingmetals, metal oxides, polymers, and so forth, that are amenable tomultiplexed synthesis.

[0107] More generally, multiplexed synthesis of nanoparticles need notbe confined to electrochemical deposition into a host. For example, thematerials described herein could likewise be prepared by sequentialevaporation, or by sequential chemical reaction. This expands thepossibilities for multiplexed nanoparticle synthesis to include alloxides, semiconductors, and metals.

[0108] Independent of the synthetic approach used, when synthesis isdone in a membrane a final critical step is required to separate eachunique type of nanorod and release all the nanorods into solution, forsurface preparation or denaturation. In the preferred embodiments of theinvention this is done by chemical dissolution of the membrane andelectrode backing, using a series of solvents. These solvents could beacids, bases, organic or aqueous solutions, at one or more temperatureor pressures, with one or more treatment times. Two additional releasetechniques are: (i) Following synthesis, whether on membrane or planarsubstrate, die separation techniques from the semiconductor industry canbe utilized. The substrate will be mated to a flexible adhesivematerial. A dicing saw cuts through the substrate, leaving the adhesiveintact. The adhesive is then uniformly stretched to provide physicalseparation between each island, each of which is then picked upautomatically by robot and placed into a separate microwell. Anautomated fluidics station is used to introduce the necessary etchingsolutions to release each rod into solution. (ii) An alternativeembodiment is a matching microwell substrate that contains wells in thesame pattern as the individual islands in the membrane, and a matchingarray of channels through which flow etching solutions. The membrane orwafer can be sandwiched between the microwell substrate and the channelarray. Etching fluid is then introduced into the channels whichdissolves the Ag backing and carries the nanorods into the correspondingwell. Other means for removing the particles from the membrane are alsopossible, including but not limited to laser ablation, heating, cooling,and other physical methods.

[0109] The membrane-based template-directed synthesis techniques arepreferred because they are capable of making a very large number of verysmall nanorods. The electroplating conditions can be adequatelycontrolled to produce many types of nanorod bar codes. For applicationssuch as multiplexed immunoassays, where tens to many hundreds of typesare required, known techniques are adequate and can simply be scaled upto provide the necessary number. For applications such as proteomicsignatures, where from dozens to many thousands of types are required,higher throughput synthesis techniques and the ability to uniquelyidentify each of thousands of different bar codes are required.

EXAMPLES

[0110] The following examples are provided to allow those skilled in theart access to information regarding various embodiments of the presentinvention, and are not intended in any way to limit the scope of theinvention.

Example 1

[0111] One embodiment of the present invention is directed to thetemplate-directed synthesis of multiple flavors of nanobar codes for thepurpose of multiplexed assays. For this application it is desirable toconstruct a variety of different flavors which are easily distinguishedby optical microscopy. For example, 10 different flavors of nanobarcodes were individually synthesized according to the table below, usinggold and silver segments. Note that the description field of the tableindicates the composition of each nanobar code by segment material andlength (in microns) in parentheses. For example, Flavor #1 is 4 micronslong gold, and Flavor #2 is 2 microns gold followed by 1 micron silver,followed by 2 microns gold. Flavor # Description # Segments Length 1 Au(4) 1 4 μm 2 Au (2), Ag(1), Au(2) 3 5 μm 3 Au(1), Ag(1), Au(1), Ag(1), 55 μm Au(1) 4 Au(2), Ag(2) 2 4 μm 5 Ag(1), Au(1), Ag(1), Au(1), 5 5 μmAg(1) 6 Ag(1), Au(4) 2 5 μm 7 Ag(4) 1 4 μm 8 Ag(1), Au(2), Ag(1) 3 4 μm9 Ag(1), Au(1), Ag(1), Au(2) 4 5 μm 10 Ag(2), Au(1), Ag(1), Au(1) 4 5 μm

[0112] A detailed description of the synthesis of Flavor #4 follows.(All other flavors were synthesized by minor and obvious changes to thisprotocol.)

[0113] 25 mm diameter Whatman Anopore disks with 200 nm diameter poreswere used for template directed nanobar code synthesis. Electrochemicalmetal deposition was carried out using commercially available gold(Technic Orotemp 24), and silver (Technic ACR 1025 SilverStreak Bath)plating solutions. All of the electroplating steps described below werecarried out in an electrochemical cell immersed in a sonication bath,which was temperature controlled to 25° C.

[0114] The synthesis of nanobar code Flavor #4 was carried out asfollows. The membrane was pretreated by evaporating ˜500 nm of silver onits branched side. To completely fill the pores on this side,approximately 1 C of silver was electroplated onto the evaporatedsilver, using 1.7 mA of plating current for approximately 15 minutes.Then an additional 1 C of silver was electroplated into the pores of themembrane from the side opposite the evaporated silver, using 1.7 mA ofplating current for approximately 15 minutes. This silver layer is usedto fill up the several micron thick “branched-pore” region of themembrane. The silver plating solution was removed by serial dilutionswith water, and was replaced by the gold plating solution. The 2 micronlong gold segments were then deposited using 1.7 mA of plating currentfor approximately 30 minutes. The gold plating solution was removed byserial dilutions with water, and was replaced by the silver platingsolution. The final 2 micron long silver segment was then depositedusing 1.7 mA of plating current for approximately 30 minutes. Themembrane was removed from the apparatus, and the evaporated silver layer(and the electrodeposited silver in the branched pores) was removed bydissolution in 6 M nitric acid, being careful to expose only thebranched-pore side of the membrane to the acid. After this step, thenanobar codes were released from the alumina membrane by dissolving themembrane in 0.5 M NaOH. The resulting suspension of nanobar codes werethen repeatedly centrifuged and washed with water.

Example 2

[0115] It is an important goal to demonstrate the ability to use a widenumber of materials in the nanobar codes of the present invention. Todate, rod structures formed by electrochemical deposition into amembrane template (alumina or track etch polycarbonate) include Ag, Au,Pt, Pd, Cu, Ni, CdSe, and Co. Primarily, the 200-nm pore diameteralumina membranes have been used for convenience. Many of the materialsare now also being used in the smaller diameter polycarbonate membranes.

[0116] CdSe is currently plated via a potential sweep method from asolution of CdSO₄ and SeO₂. Mechanical stability problems have beenencountered with the metal:CdSe interface; i.e. they break whensonicated during the process of removing them from the membrane. Thishas been remedied with the addition of a 1,6-hexanedithiol layer betweeneach surface.

[0117] The Cu and Ni are plated using a commercially available platingsolution. By running under similar conditions as the Ag and Ausolutions, it was found that these metals plate at roughly the samerate, ˜3 μm/hr. The Co is plated from a CoSO₄/Citrate solution. Theserods seems to grow fairly monodispersely, however they growcomparatively slowly, ˜1.5 μm/hr.

Example 3

[0118] One embodiment of the present invention is directed to thetemplate-directed synthesis of nanoscale electronic devices, inparticular diodes. One approach, combines the membrane replicationelectrochemical plating of rod-shaped metal electrodes with theelectroless layer-by-layer self-assembly of nanoparticlesemiconductor/polymer films sandwiched between the electrodes. Describedbelow, is the wet layer-by-layer self-assembly of multilayerTiO₂/polyaniline film on the top of a metal nanorod inside 200 nm poresof an alumina membrane.

[0119] 1. Materials

[0120] 200 nm pore diameter Whatman Anoporedisks (Al₂O₃-membranes) wereused for template directed diode synthesis. Electrochemical metaldeposition was carried out using commercially available gold (TechnicOrotemp 24), platinum (Technic TP), and silver plating solutions.Titanium tetraisopropoxide[Ti(ipro)₄], mercaptoethylaminehydrochloride(MEA),ethyltriethoxy silane, chlorotrimethyl silane werepurchased from Aldrich. All the reagents were used without furtherpurification. All other chemicals were reagent grade and obtained fromcommercial sources.

[0121] TiO₂ colloid was prepared as follows. Ti(ipro)₄ was dissolved in2-methoxyethanol under cooling and stirring. The solution was kept understirring until it became slightly yellow, after which another portion of2-methoxyethanol containing HCl was added. The molar ratio of thecomponents in the prepared solution wasTi(ipro)₄:HCl:2-metoxyethanol=1:0.2:20. This solution was diluted withwater to adjust TiO₂ concentration to 1% and allowed to age during 3weeks. The resulting opalescent sol was subjected to the rotaryevaporation at 60° C. to give shiny powder of xerogel containing 75%(w/w) titania. This xerogel was used as a precursor for the preparationof stock aqueous TiO₂ sol with TiO₂ concentration of 2.3% wt (0.29 M)and pH=3, which was stable during several weeks. XRD investigations ofthe titania xerogel allowed estimating average size of the colloidalanatase crystals at 6 nm, TEM image of the stock TiO₂ sol showsparticles of 4-13 nm in diameter.

[0122] The emmeraldine base (EB) form of polyaniline (PAN) was alsoprepared. A dark blue solution of PAN in dimethyl formamide (0.006% wt)was used as a stock solution for the film synthesis.

[0123] 2. Synthesis of Rod-Shaped Diodes

[0124] The synthesis of rod-shaped diodes was carried out as follows.Metal electrodes were grown electrochemically inside porous membrane.Briefly, the membrane was pretreated by evaporating ˜150 nm of silver onits branched side. To completely fill the pores on this side 1 C ofsilver was electroplated onto the evaporated silver. These Ag “plugs”were used as foundations onto which a bottom electrode waselectrochemically grown. The bottom gold electrode of desired length waselectroplated sonicating. The plating solution was removed by soakingthe membrane in water and drying in Ar stream. Priming the bottomelectrode surface with MEA preceded depositing multilayer TiO₂/PAN film.This was achieved by 24 hour adsorption from MEA(5%) ethanolic solution.The multilayer film was grown by repeating successive immersing themembrane in the TiO₂ aqueous solution and PAN solution in DMF for 1 h.Each adsorption step was followed by removing the excess of reagents bysoaking the membrane in several portions of an appropriate solvent (0.01M aqueous HCl or DMF) for 1 h, and drying in Ar stream. Finally, a topelectrode (Ag or Pt) of desired length was electroplated at the top ofTiO₂/PAN multilayer without sonicating. Then the evaporated silver,“plugs” and alumina membrane were removed by dissolving in 6 M nitricacid and 0.5M NaOH, respectively. (2-4 C of Au was always electroplatedon the top of Ag electrode to prevent dissolving the latter in thenitric acid. Also preliminary experiments showed that multilayerTiO₂/PAN film self-assembled on plane Au(MEA) substrate was notdestroyed in the 0.5 M NaOH.) The resulting rod-shaped diodes wererepeatedly centrifuged and washed with water.

[0125] In most of the experiments, chemical passivation ofAl₂O₃-membrane pore walls was applied using treatments with propionicacid or alkylsilane derivatives. In the latter case, a membrane wassuccessively soaked in absolute ethanol andanhydrous toluene ordichlorethane for 1 h, after which it was immersed in a ethyltriethoxysilane solution in anhydrous toluene (2.5% vol) or a chlorotrimethylsilane solution in anhydrous dichlorethane (2.5% vol) for 15 h. Then themembrane was successively soaked for 1 h in the appropriate anhydroussolvent, a mixture (1:1) of the solvent and absolute ethanol, theabsolute ethanol, and finally was dried in Ar stream. Wetting so treatedmembranes with water revealed hydrophobic properties of their externalsurface. Transmission IR spectra of the membrane treated withethyltriethoxy silane or propionic acid showed the appearance of weakbands at 2940, 2865, 2800 cm−1, which can be assigned to C—H stretchingvibrations of alkyl and alkoxy groups.

[0126] 3. Characterization

[0127] Transmission electron microscope (TEM) images were obtained witha JEOL 1200 EXII at 120 kV of accelerating voltage and 80mA of filamentcurrent.

[0128] Optical microscope (OM)images were recorded. Transmission IRspectra were recorded using a Specord M-80 CareZeiss Jena spectrometer.I-V characteristics for rod-shaped diodes were measured in air atambient temperature.

[0129] TEM images of some typical “striped” bimetallic Au/Pt/Aunanorods, grown electrochemically inside the porous alumina membrane,showed that the two rod ends differed in their topography—one of the rodends appeared to be bulging or rounded while the other rod end had anapparent hollow in the middle. Such differences in rod end appearancecould be explained by adsorption of some amount of metal ions on porewalls, promoting metal (e.g. Ag) growth in the near-wall space andcausing the hollow formation in the pore middle space. During theelectroplating of a second metal “stripe” (e.g. Au), the growing metalfollows the surface of the bottom rod and fills the hollow thus formingthe rounded end. Further rod growth results in a cup-like end due to themetal adsorption on the pore walls. Each sequential metal segment growsin the same way in the end of the underlying segment.

[0130] It is unlikely that the relatively rough surface on the top endof a rod may be completely covered with the ultrathin TiO₂/PAN film thuspreventing immediate contacts between bottom and top metal electrodes.From preliminary experiments on plane Au-substrates, it was found thatthe multilayer TiO₂/PAN films grown on smoother surfaces demonstratedbetter reproducibility in their rectifying behavior. Passivation(hydrophobization) of Al₂O₃-terminated surface of pore walls withpropionic acid or alkylsilane derivatives, such as ethyltriethoxysilaneor chlorotrimethyl silane, was tried to smooth down the top rodend surface by reducing the metal adsorption on the pore walls. Thehydrophobization of pore walls may also be expected to prevent TiO₂particles from adsorption on the wall surface rather than on metalelectrode surface situated in the depth (˜65 μm) of the pore. It wasshown that the TiO₂ particles readily formed a densely packed layer on aplane Al/Al₂O₃ substrate. A typical higher resolution image of rod'supper part confirmed that the cup-like ends are situated at the top ofthe rods, and showed that the wall passivation to some extent resultedin smoothing of the surface of rod ends.

[0131] An optical micrograph of Au/(TiO₂/PAN)₁₀/Ag/Au rods, preparedusing the membrane derivatized with ethyltriethoxy silane, showednanorods of uniform length, in which a silver segment is clearly seenbetween two gold ends. TEM images of such a rod, recorded in the firstseveral seconds, revealed no visible signs of a metal/film/metalheterojunction within the rod. However, after focusing the electron beamon this rod for some time (typically tens of seconds), a break appearedin the rod and metal segments became separated, perhaps due tobeam-induced metal melting, in the neighborhood of the Au/film/Agheterojunction. In higher resolution TEM images of this break, particlesof 5-10 nm in diameter, which adhere to both metal ends, were observed.Apparently, TiO₂ nanoparticles are present between two electroplatedmetals. The OM and TEM data suggest that the self-assembly of multilayerTiO₂/PAN film on the Au rod top can be realized inside the membranepores, and that the self-assembled film does not prevent Ag rodelectroplating on the top of the film. It should be noted that TEMimages in all likelihood do not give a true picture of the multilayerTiO₂/PAN film inside the rod because of high probability of themechanical film destruction while separating partially melted metal rodends. Longer time exposure of the rod to the electron beam causescomplete destruction of the heterojunction and arising two individualnanorods with nanoparticles stuck to their ends.

[0132] In order to investigate multilayer TiO₂/PAN film sandwichedbetween Au and Ag rods, Au/(TiO₂/PAN)r/Ag nanorods were prepared andtheir top Ag electrode was dissolved in nitric acid: The remaining 2C Aurods with (TiO₂/PAN)₆ film deposited on their top were analyzed by TEM.Preliminary studies showed that ellipsometric thickness of multilayerTiO₂/PAN film self-assembled on plane Au(MEA) substrate did not decreaseafter immersion in 6 M HNO₃ for 30 min suggesting stability of the filmin the acidic medium. Furthermore, similar to the Au/(TiO₂/PAN)₁₀/Ag/Aurods described above, TEM image of the Au/(TiO₂/PAN)₆ rod taken in thefirst several seconds did not reveal any particles. However, duringlonger exposure to the electron beam, gold melted revealing nanoparticlefilm on the rod's top. It can be seen that the upper contour line of thefilm is very close to that of Au rod before melting. This fact isconsistent with the cup-shaped top of the metal rods. The multilayerfilm grows on the surface both of cup bottom and cup walls andapproximately retains cup shape after the thin walls have melted. Thisexplanation is consistent with observed film height of ˜100 nm, whichallows estimating rather gold cup depth than (TiO₂/PAN)₆ film thickness.Ellipsometric thickness of TiO₂/PAN)₆ film self-assembled on a planeAu(MEA) substrate is estimated at about 10 nm.

[0133] I-V characteristic of the Pt/(TiO₂/PAN)₃ TiO₂/Au rod-shapeddevice reveals current rectifying behavior. The forward and reverse biasturn-on potentials are ˜−0.2 and ˜0.9 V, respectively.

Example 4

[0134] Segmented nanoparticles can be synthesized from membranesproduced using photolithographic techniques as follows: A silicon waferis spin coated with a the photoresist AZ®4620 (Clariant Corp.,Somerville, N.J.). The spin coating is conducted at 1000 rpm for 40seconds. The photoresist-coated substrate is then baked for about 200seconds on a hot plate coater at 110° C. Outgassing to remove volatilematerials is conducted by allowing the material to sit out at roomtemperature for at least 24 hours. The photoresist-coated substrate isthen exposed to radiation for about 3100 msec using a mask to patternthe resist. The photoresist is then developed using AZ®400K (potassiumbased buffered developer) (Clariant Corp.) for about 15 minutes toreveal cylindrical pores. The porous membrane is spin rinsed and dried.The membrane is then evacuated for about an hour using a high vacuum.

[0135] The pores of the membrane can be filled with alternating bands ofmetals to form segmented nanoparticles as follows: copper is plated intothe pores using 1.0 mA for about 5 minutes. Gold is plated into thepores using 1.0 mA for about 120 minutes. Silver is plated into thepores using 1.0 mA for about 30 minutes. Gold is again plated into thepores using 1.0 mA for about 30 minutes. The photoresist is thendissolved using acetone. An SEM (top view) of the template after platingis complete and the photoresist has been dissolved is shown in FIG. 4.The tops of the nanoparticles, having diameter approximately 2.5 to 3 μmare visible. The segmented nanoparticles may then be separated from themembrane using acetic acid for about 60 minutes.

Example 5

[0136] In another embodiment, segmented nanoparticles can be synthesizedfrom membranes produced using photolithographic techniques as follows: Asilicon wafer is initially sputtered with chromium and then gold, toform layers of about 200 Å and 1000 Å in thickness, respectively. Awafer singe is then performed to remove water vapor and/or residualorganics by heating at a temperature of about 150° C. for 30 minutes.The conductive substrate is then spin coated first with 50%hexamethyldisilazane (HMDS), and then with SPR®220 (Shipley Corp.,Marlborough, Mass.). The spin coating is conducted at 3500 rpm for about40 seconds. The photoresist-coated stack is then baked for about 200seconds at 90° C. on an SVG coater (Silicon Valley Group, San Jose,Calif.). This “soft bake” step is recognized in the art to reducesolvent concentrations in the photoresist and improve adhesion byrelieving film stresses. Outgassing to remove volatile materials is thenconducted by allowing the material to sit out at room temperature for atleast 24 hours. The photoresist-coated stack is exposed to radiation forabout 1600 msec using an appropriate mask to pattern the surface. Thephotoresist is developed using LDD26W for about 100 seconds. Thedeveloping step is repeated three times to reveal cylindrical pores.Oxygen plasma is applied at 65 W for about 4 minutes, removing anyphotoresist debris. The porous membrane is evacuated for about an hourusing high vacuum.

[0137] The pores of the membrane can be filled with alternating bands ofmetals to form nanoparticles as follows: copper is plated into the poresusing 1.0 mA for about 5 minutes. Gold is plated into the pores using1.0 mA for about 115 minutes. After plating is complete, the photoresistis dissolved using acetone. The segmented particles are separated fromthe substrate using acetic acid for about 60 minutes. An SEM (side view)of a nanoparticle made according to the above procedure is shown in FIG.5.

Example 6

[0138] In another embodiment, segmented nanoparticles can be synthesizedfrom membranes produced using photolithographic techniques as follows:Cr and then Au, are sputtered onto a silicon wafer to a thickness ofabout 200 Å and about 1000 Å, respectively. The wafer is heated at atemperature of about 150° C. for 30 minutes and then spin coated with50% HMDS to promote adhesion. The stack is then spin coated withpolyimide, to a thickness of about 10 μm followed by a soft bake toremove solvent. Aluminum is then sputtered on the stack to form an etchstop layer of about 3000 Å in thickness. After spin coating again with50% HMDS, the stack is spin coated with Shipley 3612 (5,500 rpm for 30min.) resulting in a layer about 1 μm in thickness, and then heated at atemperature of about 90 ° C. for about 60 seconds. Outgassing to removevolatile materials is conducted by allowing the material to sit out atroom temperature for at least 24 hours.

[0139] The stack is exposed to radiation which has traveled through amask to transfer a pattern which will leave cylindrical holes in thephotoresist. The photoresist is developed using LDD26W for 60 seconds.Deep reactive ion etching is used to transfer the pattern in thephotoresist to the aluminum etch stop (450 W, 200 mTorr, 60 sec. BCl₃ 40sccm, Cl₂ 30 sccm, N₂ 40 sccm). The stack is then placed in water toremove any residual choloride, and then spin rinsed and dried. Thepolyimide is then etched via deep reactive ion etching using oxygenplasma (500 W. 250 mTorr, 300 sec., O₂ 50 sccm) to reveal cylindricalpores in the polyimide. The resulting membrane is evacuated for one hourusing high vacuum. An SEM (cross-sectional view) of a template preparedaccording to the above procedure is shown in FIG. 6.

[0140] The cylindrical pores can subsequently be filled with alternatingbands of metals as set forth described above.

We claim:
 1. A method for the manufacture of a free standingnanoparticle, the method comprising: a. providing a planer substrate; b.causing deposition of a layer of material on said surface; c. placing amask over said material coated substrate, said mask comprising aplurality of cylindrical posts and prepared by the method comprising: i)providing a resist-coated substrate; ii) exposing a pattern on saidresist-coated substrate using a photolithographic technique; iii)developing said pattern; and iv) etching said pattern; d. releasing saidnanoparticles from the surface of said substrate.